Determination of SMN1 and SMN2 copy number
High quality genomic DNA was extracted from the peripheral blood of the patients (QuickGene DNA Whole Blood Kit L, Kurabo, Osaka, Japan) according to the manufacturer’s instructions.
Homozygous deletion of the SMN1 gene was searched for using ACRS (Amplification Created Restriction Site; (van der Steege et al. 1995). SMN1 and SMN2 copy numbers were determined by using two QMPSF assays as previously described (Quantitative Multiplex PCR of Short fluorescent Fragments; (Saugier-Veber et al. 2001; Vezain et al. 2010). The QMPSF-DraI specifically introduces a DraI restriction site into SMN2 amplicons but not into SMN1 amplicons whereas QMPSF-HinfI creates a HinfI restriction site into SMN1 amplicons but not into SMN2 amplicons. PCR products were digested with DraI or HinfI, separated by electrophoresis and analyzed on an ABI Prism 3130 xl Genetic Analyser instrument (Applied Biosystems, Foster City, CA).
Approximately 3 μg of high quality DNA was sheared with a Covaris E220 DNA Sonicator (Covaris, Inc., Woburn, MA, USA), coding regions and intron-exon boundaries (±100bp) were captured using a SureSelectXT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The enriched libraries were sequenced on a NextSeq500 system (Illumina, Inc., San Diego, CA, USA) with 2x150 bp paired-end reads. Image analysis was performed by Real-Time Analysis (RTA 188.8.131.52) and the bcl2fastq conversion software (Illumina, v2.20) was used for reads demultiplexing and generation of Fastq files. Sequenced reads were mapped to the human reference sequence (GRCh37, Hg19) with the Burrows-Wheeler Aligner (BWA v.0.7.17). Read duplicates were marked with Picard tools (v2.18.0), local realignments around indels and base-quality-score recalibration were performed with the Genome Analysis Toolkit (GATK 184.108.40.206). Single-nucleotide variants and small indels were identified with the GATK HaplotypeCaller and were filtered according to the Broad Institute’s best-practice guidelines (Table S1). Variants were then annotated with SnpSift (v.4.2). Mapped reads were visualized using Alamut visual software (v.2.11, Sophia Genetics, St Sulpice, Switzerland).
SMN1 specific genomic insertion validation
Specific primers were used to PCR amplify SMN1-SVA (SMN1Int6-44G-F, SMNInt7-SVA-R) or SMN2-SVA (SMN2Int6-44A-F, SMNInt7-SVA-R) DNA fragment. PCR fragments were resolved on 1.5% agarose gel. PCR primers are listed in Table S2 and protocol used is available upon request.
DNA Sequencing of the SMN1 intron 7-SVA-F Region of Interest
Specific SMN1-SVA DNA fragment was long-range PCR amplified using a forward primer at SMN intron 7-SVA boundary (SMNInt7-SVA-F) and a reverse primer specific to SMN1 c*3+215 (SMN1Int7-215A-R). PCR fragment was resolved on 1% agarose gel and purified. We sequenced in a walk along the chromosome to get closer into the inserted SVA fragment (SVA-1F to SVA-9F). Sanger-sequencing reactions were carried out with BigDye™ Terminator v3.1 cycle sequencing kit and a 3500 Series Genetic Analyzer (Applied Biosystems). PCR and sequencing primers are listed in Table S2 and protocols used are available upon request. Description is based on the SMN1 reference sequence NM_000344.3.
Quantification of SMN1 and SMN2 full-length transcript by using SNaPshot assay
Blood samples were collected using the PAXgeneTM Blood RNA Tube system (Qiagen, Courtaboeuf, France) and RNA was extracted with the PAXgeneTM Blood RNA Kit (Qiagen). Total RNA (100ng) was reverse-transcribed into cDNA using Verso cDNA kit (ThermoFisher Scientific, Waltham, USA) together with a mix of anchored oligo-dT primers and random hexamers (3:1). cDNA were PCR-amplified under quantitative conditions using the forward primer at the junction of SMN exon 4 and 5 (SMNEx4-5F) and the reverse primer in exon 7 (SMNEx7-R). Genomic DNA from the same individual was amplified, under quantitative conditions, by using the forward primer in SMN intron 6 (SMNInt6-F) and the same reverse primer in exon 7. Purified PCR products (20ng) were used as templates in SMN exon 7 c.840 specific primer extension reactions (0.2µM; SMNEx7-840-PE-R) with the SNaPshotTM Multiplex Kit (Applied Biosystems), according to the manufacturer’s instructions. Then, the reaction products were treated with Shrimp Alkaline Phosphatase (1U; Roche Diagnostics, Meylan, France), and separated on a 3500 Series Genetic Analyzer (Applied Biosystems). Primer extension products were quantified by measuring the area of the peaks corresponding to each allele. Given the unequal fluorescence intensities of the incorporated fluorophore-labeled dideoxynucleotides, the relative amount (in percentage) of wild-type and mutant FL-SMN transcripts produced by each SMN gene was determined by normalizing the cDNA results to the matching gDNA data. PCR and extension primers are listed in Table S2. Experiments were performed in triplicate.
Lymphoblastoid Cell Lines (LCL) derived from patient and controls were maintained in RPMI 1640 medium (GIBCO; Life Technologies, Carlsbad, California, USA) with 10% fetal calf serum (Invitrogen, ThermoFisher Scientific) and 1% L-glutamine (Invitrogen) at 37°C with 5% CO2.
Total RNA was extracted from LCLs using the NucleoSpin RNA kit, according to the manufacturer’s protocol (Macherey Nagel, Hoerdt, France). Total RNA was also extracted from patient LCL after puromycin traitment (10 μg/mL puromycin is added to the culture 5.5 h before harvesting). A second DNase traitment was performed to ensure RNA specific sequencing (AMPD1, Sigma-Aldrich, St Louis, MO). The yield and quality of the isolated RNAs were assessed using a NanoDrop 2000 (Thermo Fisher Scientific) and a TapeStation 4200 (Agilent Technologies), respectively. rRNA were depleted from 1µg of high-quality total RNA (RIN>9) using the NEBNext Ribosomal Depletion kit (New England Biolabs, Ipswich, MA, USA). cDNA libraries were then prepared from each rRNA depleted RNA samples using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina according to the manufacturer’s instructions (New England Biolabs). Total RNA-seq libraries were sequenced on a NextSeq 500 platform (Illumina) using high-output paired-end sequencing (2 × 75 bases). About 90 million reads were generated from each library with a mean quality scores above 30 (Table S3). Reads demultiplexing and generation of Fastq files were obtained using bcl2fastq conversion software (Illumina, v2.20). The bioinformatics data analysis was performed using the nf-core RNASeq analysis pipeline (v3.1, (Ewels et al. 2020). RNA-Seq reads were aligned to the human reference genome (GRCH37, hg19) using STAR aligner (v2.6.1d). Bam files visualization and read count determination were performed using Integrative Genome Viewer (IGV v2.5.2, Broad Institute).
RNA Sequencing of the SMN1-SVA isoform
Total RNA obtained from LCLs was reverse-transcribed into cDNA using Verso cDNA kit (anchored oligo-dT and random hexamers primers). SMN-SVA cDNA fragment was PCR amplified using a forward primer at SMN exon 4-5 boundary (SMNEx4-5F) and a reverse primer in SMN exon 8 (SMNEx8-R). PCR fragments were resolved on 1% agarose gel and purified. To elucidate the sequence of the isoform, we conducted consecutive Sanger sequencing in a walk along the chromosome using SVA-1F to SVA-9F primers. PCR and sequencing primers are listed in Table S2 and protocols used are available upon request.